Type IV pili are produced by many Gram-negative bacteria of medical and veterinary importance. The biogenesis of type IV pili appears to be quite complex, but is poorly understood. Because all of the genes required for the biogenesis of the bundle-forming pilus (BFP) type IV fimbriae of enteropathogenic E. coli (EPEC) are known, this system serves as an excellent model to dissect this process. The central hypothesis of this proposal states that BFP are assembled by a multi-component Bfp machine that is made up of the products of the genes of the bfp operon. The investigators plan to further their studies of BFP in an effort to progress toward a complete understanding of the type IV pilus assembly machine. Because of the complexity of the type IV pilus biogenesis machinery, they find it convenient to conceptualize the whole as consisting of two subassemblies, which they intend to define. To those ends, they propose four specific aims. First, they will determine whether each Bfp protein is located in the cytoplasm, periplasm, inner or outer membrane. This information is needed to refine hypothesis regarding which Bfp proteins directly interact. Second, they will describe the architecture of the Bfp outer membrane subassembly. Evidence from their lab and other labs suggests that BfpB, BfpG and BfpU interact with each other in the outer membrane. Since they have already developed a functional, purified histidine-tagged BfpU fusion protein, they will use BfpU as a tool to define the interactions among these three proteins. Third, they will describe the architecture of the Bfp inner membrane assembly. They have examined the topology of the BfpE protein and found that it contains three transmembrane domains that span the inner membrane. They will use this knowledge as a basis for investigating interactions among BfpE, BfpC, BfpD and BfpF. Fourth, they will identify the proteins that link the inner and outer membrane subassemblies of the Bfp machine. One hypothesis states that BfpI, BfpJ, BfpK and BfpL serve this function. As data are gathered from specific aims 1-3 they will modify and test this hypothesis. From the results of these studies a clearer picture of the overall design of the type IV pilus biogenesis machine will emerge. Further studies including determining binding constants and atomic structure will be required to reveal how the machine functions to assemble these important virulence factors.